The complexity of the ubiquitin system requires advanced approaches/methodologies to identify ubiquitinated substrates, uncover diversity in targeting mechanisms, and elucidate pathways that control downstream steps. From profiling ubiquitin-protein conjugates levels on a proteome scale to the quantitative analysis of individual polyubiquitin chains, mass spectrometry is making critical advances in ubiquitin biology. These advances illuminate other important unsolved issues. For example, although ubiquitin modification sites have been identified for some proteins, for most ubiquitin substrates, lysine-acceptor sites are not yet known. Awareness of these sites provides both concrete evidence of modification and the opportunity for targeted studies. Many groups have observed that more often than not, multiple lysines within the same substrate are modified. Since a single polyubiquitin chain represents a competent degradation signal, the purpose of the additional complexity is unclear. Part of this regulatory complexity is likely attributable to the roles of several prominent proteasome-associated ubiquitin receptors, including Dsk2, Rad23, and Ddi1, commonly believed to modulated substrate recognition. To uncover the mechanism of substrate-specific regulation by these receptor proteins, the subsets of conjugates whose degradation is dependent on each will need to be identified. This proposal details how we will develop a novel strategy to determine ubiquitination sites en masse. In addition, we propose to extend our current methods and precisely quantify the amount of ubiquitination occurring on individual substrate lysines enabling hypothesis-driven experiments in vivo and in vitro in a substrate-specific manner. Finally, we will combine stable isotope labeling with shotgun proteomics to identify and differentiate substrates for yeast ubiquitin receptor proteins (UBL/UBA-containing) including Rad23, Dsk2, and Ddi1, setting the stage for similar endeavors in mammalian cells. Together, these integrated proteomics strategies will provide new insights concerning the role of ubiquitin receptor proteins and their influence on polyubiquitin chain synthesis, structure, and function for physiologically relevant ubiquitin substrates. PUBLIC HEALTH RELEVANCE The modification of a protein by ubiquitin attachment produces a signal utilized with cells for a variety of purposes. The most studied outcome of a ubiquitin-conjugated protein is the degradation (proteolysis) of the protein. However, nonproteolytic eventualities are also the subject of intense research efforts. Nearly every aspect of research within the ubiquitin system shows great promise for the improvement of human health. In addition to its critical role in normal physiology, malfunctions in protein ubiquitination have been implicated as a contributing factor in the causation of many diseases and syndromes as diverse as cancer and Alzheimer's disease. This proposal will provide new technologies to measure critical end points in ubiquitin biology including the complex structure and function of polyubiquitin (multiple ubiquitination) chain formation and the discovery of substrates for ubiquitinated protein receptors.